Biosynthesis of Chiral Amino Alcohols via an Engineered Amine Dehydrogenase in E. coli.

Feifei Tong,Zongmin Qin,Hongyue Wang,Hui Ming,Yingying Jiang,Zhoutong Sun,Junkuan Li,Yazhong Xiao,Ge Qu

doi:10.3389/fbioe.2021.778584

Abstract

Chiral amino alcohols are prevalent synthons in pharmaceuticals and synthetic bioactive compounds. The efficient synthesis of chiral amino alcohols using ammonia as the sole amino donor under mild conditions is highly desired and challenging in organic chemistry and biotechnology. Our previous work explored a panel of engineered amine dehydrogenases (AmDHs) derived from amino acid dehydrogenase (AADH), enabling the one-step synthesis of chiral amino alcohols via the asymmetric reductive amination of α-hydroxy ketones. Although the AmDH-directed asymmetric reduction is in a high stereoselective manner, the activity is yet fully excavated. Herein, an engineered AmDH derived from a leucine dehydrogenase from Sporosarcina psychrophila (SpAmDH) was recruited as the starting enzyme, and the combinatorial active-site saturation test/iterative saturation mutagenesis (CAST/ISM) strategy was applied to improve the activity. After three rounds of mutagenesis in an iterative fashion, the best variant wh84 was obtained and proved to be effective in the asymmetric reductive amination of 1-hydroxy-2-butanone with 4-fold improvements in k cat /K m and total turnover number (TTN) values compared to those of the starting enzyme, while maintaining high enantioselectivity (ee >99%) and thermostability (T 50 15 >53°C). In preparative-scale reaction, the conversion of 100 and 200 mM 1-hydroxy-2-butanone catalyzed by wh84 was up to 91–99%. Insights into the source of an enhanced activity were gained by the computational analysis. Our work expands the catalytic repertoire and toolbox of AmDHs.

Highlights

As essential structural moieties, chiral α-amino alcohols are widely applied to produce synthetic and natural bioactive molecules (Ager et al, 1996; Erlanson et al, 2011)
Based on the crystallographic structure of SpLeuDH (PDB ID: 3VPX, Zhao et al, 2012), this SpAmDH variant (K68S/N261L, dubbed wh18) showed excellent stereoselectivity (>99% ee) in the transformation of 1a to 1b, while the catalytic efficiency was modest when it was applied in large-scale production (Wang et al, 2020)
The combinatorial active-site saturation test (CAST)/iterative saturation mutagenesis (ISM) strategy was employed with the aim of enhancing the activity of SpAmDH

Summary

Introduction

Chiral α-amino alcohols are widely applied to produce synthetic and natural bioactive molecules (Ager et al, 1996; Erlanson et al, 2011). Direct asymmetric reductive amination of ketones with free ammonia to produce chiral amino alcohols is a highly aspirational transformation (Abrahamson et al, 2013; Pushpanath et al, 2017). SCHEME 1 | SpAmDH-catalyzed asymmetric reductive amination of 1a to (S)-1b by using ammonia as the sole amino donor, and glucose dehydrogenase (GDH) cell-free extract (CFE) for NADH cofactor regeneration. Enzymes as catalysts are increasingly explored as essential tools in asymmetric reductive aminations (Reetz, 2011; Abrahamson et al, 2013; Schrittwieser et al, 2015; Chen and Arnold, 2020; Winkler et al, 2021). Native amine dehydrogenases (AmDHs) have been identified that they can directly utilize ammonia as a sole amino source in reductive amination but with insufficient enantioselectivity (Itoh et al, 2000; Mayol et al, 2019)

Methods

Results

Conclusion